Liquid Scintillation Phosphor

Hello everyone!

I am doing some polymerase assays with monoclonal antibodies
inhibition. That is why i want to utilize Scintillation. Can anybody
share knowledge on this procedure, i mean protocols...

I am precipitationg RNA with Salmon Sperm 100 microgram (adding to 40
microliters reaction 10 microliters).. then i add 1 ml of 10% TCA with
Na4P2O7 (some 1%).. waiting for 30 min on ice.. then filtering samples
through GlassFiber GF/C... and washing it with the same 10% TCA.. and
EtOH...

Then just soak into scintillation medium...

1) How long should i wait till i place my tubes into counter??? (i
mean i typically wait for some 10 min)

2) Can you explain what actually happens with all those flashes?
beta-electrons kick some double bonds? or something? then we have
somekind of photochemical reaction (something like in our eyes with
retinal)....I would like to understand that more

3)Any additional info is highly appreciated


Thanx!

----- Drew

redeamer wrote:



The most sensitive way to detect and quantitate beta-radiation is to
measure the luminescence that is created when beta-radiation interacts
with certain chemicals (fluors). Toluene, xylene and other aromatic
hydrocarbons give off ultraviolet light when hit by beta-rays. This
far-UV light is used to generate near-UV fluorescent light in PPO
(2,5-Diphenyloxazole), which in turn induces blue fluorescence in POPOP
(1,4-Bis-2-(5-phenyl-2-oxazolyl)-benzene). Szintillators for aqueous
samples also contain Triton-X100 as emulifier, so that a homogeneous
mixture is obtained. If ZnCl2 is added to the szintillator, counting of
gamma-rays is also possible (interaction of gamma-rays with Zn ions
produces Compton electrons, which can be counted like beta-rays).
Efficiency in this case depends on gamma-ray energy, (80 % for 125I, 15
% for 22Na). In any case, efficiencies are not much lower than with a
Na(Tl)I-crystal gamma-counter, so if you ever have to choose between
buying a gamma- or beta-counter, get the beta.

The blue light is detected with photomultipliers or solid-state
detectors. Since each photomultiplier produces some noise signal,
beta-counters use 2 photomultipliers both facing the sample vial. The
signals from these tubes are send through a coincidence detector, a
count is registered only if both tubes produce a signal at the same
time. This lowers noise to about 30-50 events per minute.

The intensity of the light flash is proportional to the energy of the
beta-ray causing it, hence it is possible to distinguish high (32P),
medium (14C, 35S, 33P) and low energy (3H) radiators, and even count
them separately in the same sample. Counting efficiency is better than
90% for mediuum and high energy isotopes, but significantly lower for
weak radiators like 3H (usually about 30 %).

Samples should be of neutral pH, as both high and low pH can induce
chemiluminescence in scintillation cocktails, which is difficult to
distinguish from the signal caused by beta-radiation. Chemiluminescence
is also reduced by low temperatures (about 10 Celsius). Phosphorescence
is another possible problem, samples should be stored under reduced
light for about 1 h before counting. However, in every counter I have
ever worked with this is ensured by the usual backlog of samples.

Since you are working with samples on filters, you have to make sure
that the filter is soaked with szintillator befor counting, that is the
filter should be translucent.

That was the quick answer, but you should realy get some training to
make efficient use of a beta-counter. Usually universities offer brief
(about a week) practical classes as introduction to this topic.

Thank You, Dr Engelbert Buxbaum,

for your really full answer to my question.

But now I have another problem with sticking the RNA to my filters. I
have done it as i wrote in initial post: vacuum pump, pre-wetted the
filter with 10% TCA, then spotted (filtrated) my probe onto it, etc...
I have a feeling that majority of my probe somehow does not stick to
this GF/C filter.

May be I should reallly do as in one of the Duncan Clark posts: spot
onto dry filter, wait until it dries, then wash in 0.1 M HCL, and 5mM
Na4P2O7 and then briefly in methanol. Then dry and add szintillator.

Does this procedure suit other filters too? like DE 80 or something
like that? It should fix the RNA somehow, to this filter, otherwise it
will just be washed out.

I think I will actually do it like that tomorrow.


_-=//// Thank YOU \\\\=-_

---> Drew